Phytochrome enhances nitrate reductase activity in ...

Phytochrome enhances nitrate reductase activity in the lichen Evernia prunastri

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A. AVALOSAND C. VICENTE' Department of Plant Physiology, Faculty of Biology, Complutense University, 28040 Madrid, Spain Received September 14, 1984 AVALOS,A,, and C. VICENTE.1985. Phytochrome enhances nitrate reductase activity in the lichen Evernia prunastri. Can. J. Bot. 63: 1350- 1354. The lichen Evernia prunastri shortly after collection shows low nitrate reductase activity. The enzyme is induced by nitrate in the dark and this effect is enhanced by red light, the action of which is reversed by far-red light. Nitrate reductase is located in both symbionts, but mycobiont cells are responsible for the increase of enzyme activity in whole thalli floated on nitrate in the dark. When this increase is achieved in the light, it can be related to the Prr content of the photobiont cells, whereas changes in the activity of the fungal enzyme are negligible. AVALOS,A., et C. VICENTE.1985. Phytochrorne enhances nitrate reductase activity in the lichen Evernia prunastri. Can. J . Bot. 63: 1350- 1354. Le lichen Evernia prunastri, recernrnent rCcoltC, rnontre une activitC faible de la nitrate rkductase. L'enzyrne est induite par le nitrate i I'obscuritC, et cet effet est stirnu16 par la lumiere rouge lointain. La nitrate rCductase est prCsente dans les deux symbionts, rnais les cellules du rnycosyrnbiote sont responsables de ]'augmentation de 1'activitC syrnbiotique des thalles entikres flottant sur une solution de nitrate, i I'obscuritC. Quand I'augrnentation d'activitC est obtenue a la lurnikre, elle peut &trereliCe i la teneur en PI, dans les cellules du photobiote alors que les changernents de I'activitC de l'enzyrne du charnpignon sont nCgligeables. [Traduit par le journal]

Introduction Many events related to both growth and differentiation in green plants are regulated by the pigment phytochrome. However, there are not many studies concerning its activity in lichens. Giles (1970) examined the light-mediated action of phytochrome on aplanospore formation by the lichenized alga Trebouxia in relation to the effect of several phenolics on this process. This simplification, the use of a green alga which behaves as a photobiont instead of the lichen itself, can be acceptable since the occurrence of phytochrome in fungi has not been demonstrated (Mohr and Shropshire 1983). Many results indicate the action of phytochrome in several metabolic processes in lichens. It is known that the concentration of fumarprotocetraric acid in Cladonia rangiferina (Fahselt 1981) or usnic acid in Cladonia subtenuis (Rundel 1969) linearly increases as a function of light intensity, as well as a function of the activity of certain enzymes related to the metabolism of the latter phenol (EstCvez et al. 198 1). From this fact it can be concluded that there is something more than a mere trophic effect of the light, through photosynthesis, on the production of such phenols (Vicente et al. 1984). On the other hand, the activity of several enzymes of nitrogen metabolism in lichens decreases after light treatment (Vicente et al. 1978; Legaz and Vicente 1983; Legaz et al. 1983), although in these cases, it has always been assumed that inactivation of these enzymes is caused by the phenols accumulated by the action of light. Nitrate reductase is a well-documented phytochromemediated enzyme in higher plants. Jones and Sheard (1972) showed that nitrate reductase induction in etiolated pea terminal buds was enhanced by a pulse of red light and it was reversed by far-red light. Similar results have been reported for the enzyme from rice seedlings (Gandhi and Nair 1974), Sinapis alba cotyledons (Johnson 1976), and etiolated maize leaves (Rao et al. 1980). However, it has not been studied in lichens. In this work, we attempt to demonstrate some re'Author to whom all correspondence should be addressed.

lationship between nitrate reductase activity and phytochrome photoconversions in the lichen Evernia prunastri.

Materials and methods Evernia prunastri (L.) Ach., growing on branches of Quercus rotundifolia (L.), was collected in Valsain (Segovia, Spain), dried in air at room temperature, and then stored in polythene bags, at 7"C, in the dark, until required. Prior to the incubations, dry thalli were maintained at 26°C in the dark for 48 h. Then, samples of 1.0 g in air-dry weight were floated on 25 mL 10 rnM KNO3 in distilled water for 7 h at 26°C. Where indicated, 100 KM cycloheximide was added to the incubation medium 10 rnin before the first light pulse. All the incubations were started by only one irradiation with red light (photon flux rate, 25 ~ m o l m-' s-' at plant level) for 10 rnin followed by transfer to continuous darkness or by only one red irradiation immediately followed by 10 min far-red light at the same photon flux rate. In another series of experiments, both irradiation programs (10 min red light or 10 rnin red light plus 10 rnin far-red light) were repeated at the start of each hour throughout incubation (Fig. 1). 'The light source was a 60-W tungsten lamp. Red light was obtained by filtering white light through one layer of Cokin A 003 (Chrornofilter, France). Far-red light was obtained by filtering white light through one layer of Cokin A020 and one layer of Cokin A003. The spectral energy distribution of both kinds of light is shown in Fig. 2. Where indicated, the photon flux rate was changed to 250 kmol m - z . s-' for 10 rnin. The nitrate reductase assay was carried out by washing thallus samples with distilled water and grinding them in a mortar with 25 rnL acetone to remove lichen phenolics. This treatment is sufficient to avoid the presence of the phenols in the subsequent buffered extracts, as demonstrated by Martin-Falquina and Legaz (1984). The cellular debris was then dried in vacuo and ground with 15 rnL 0.1 M Tris-HCI containing 50 KM KCN to prevent the action of oxidases. This extraction procedure was properly effective, since nitrate reductase inhibition by cyanide requires reducing conditions (Barea et al. 1976) and does not occur on oxidized nitrate reductase. Crude extracts, buffered at pH 7.5, were centrifuged at 21 000 X g for 20 min at -2"C, and the supernatants were used for enzyme assays. Nitrate reductase activity was estimated either by following NADH oxidation at 340 nm or by measuring by diazotation the nitrite formed (Paneque and Losada 1966). When the stoichiornetry of NADH utili-

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AVALOS AND VICENTE

c A


i 0 min R

Darkness

0 -.- 0.20 In

A A

Y

10min R+lOminFR

C 3

c

.j

.E

-

0.15

d

: 7°'e +

e

/ 3fq

-

r

: ..- 0.10 -

/

Y

U

aJ 0

Er 0.05,-

20-

=.

1

O

N

1h

3

-

c

W

FIG. I. Programs of irradiation used in this work. R, red light; FR, far-red light.

0

0

I 1

I 2

X

0 1 2 3 L 5 6 7 Time (hours)

I

I

I

3 4 5 6 Time ( hours)

I 7

FIG. 3. Time course of nitrate reductase activity in Evernia prunastri thallus floated on 10 rnM KNOT in the dark at 26OC. The insert shows the time course of enzyme activity when 100 pM cycloheximide is added to the incubation media. Data are the means of three replicates. Vertical bars give standard error where it is larger than the symbols. a, y = 0.06 + 0.08. In(l + x ) , r = 0.94; 0, y = 0.0026 - 0.0003x, r = 0.83.

100-

3

increase of absorbance at 660 and 730 nm, using a dual wavelength DMS 90 Varian spectrophotometer.

800

760

720

680 h (nm)

640

600

FIG. 2. Spectral energy distribution of red and far-red light. of quantum flux at 660 to quantum flux at 730 nm.

i,ratio

zation and nitrite production was established, the first method was chosen as a more convenient, fast routine assay. Enzyme activity, as specific activity, is expressed as the decrease in an absorbance unit per milligram protein per minute (Roldan et al. 1978). Protein was estimated by the Folin-phenol reaction (Lowry et al. 1951) modified by Potty (1969) and Hartree (1972), using bovine serum albumin as a standard. Isolation of both symbionts was carried out according to Legaz and Vicente (1981) by using 2% polyvinylpirrolidone (PVP) in 0.1 M Tris-HCI buffer, pH 7.5, to remove lichen phenolics without membrane damage. Drops of photobiont and mycobiont suspensions were examined by light microscope to obtain preparations of the symbionts with least contamination. The cells were collected by centrifugation at 10 000 x g for 10 min at 4OC and the pellets were resuspended either in cyanide-containing Tris-HCI buffer, for enzyme assay, or in 10 mM N-morpholino-3-propane sulfonic acid buffer, pH 7.2, containing 0.4 M sucrose, 1 mM EDTA, and 1 mM dithiothreitol ( D n ) (Yamamoto and Furuya 1979) for phytochrome assay, in a final volume of 3 mL. Both suspensions were disrupted in a MSE sonic oscillator at 20 kcycles . s-' for 1 min with ice-cold protection. For the nitrate reductase assay, particle suspensions were centrifuged at 21 000 X g for 20 min at -2OC and the supernatants were used. Particle suspensions of algal cells were directly used for phytochrome assay according to Vicente and Garcia (1981) with 20 mg of C a C 0 3 added per millilitre of sample as scattering agent, instead of the amount (1.5 g mL-I) used by Butler and Norris (1960). Phytochrome was estimated by the

Results Fragments of dry thallus of E. prunastri, stored for 48 h in the dark at 26OC, are floated on 10 mM K N 0 3 in distilled water or in the same medium to which 100 pM cycloheximide is added. In these conditions, the time course of nitrate reductase activity is shown in Fig. 3. Enzyme activity logarithmically increases, as a function of the time, when thalli are floated on nitrate alone. Initial rate of activity increase has been estimated as about 55 p U h-I (the slope value of the asymptote in the origin), wheieas the final rate of increase is f o i n d to be 0.22 pU h-' (the slope value of the asymptote in the extreme). This increase is totally nullified after the addition of the inhibitor of translation, cycloheximide, in the incubation medium (insert in Fig. 3). Although enzyme assays were performed immediately after extraction, the stability of the enzyme was assayed since nitrate reductase from higher plants is a very unstable protein that requires the use of protective agents. However, Evernia enzyme behaves as a stable protein. Nitrate reductase was assayed before and after storage at 10°C for 24 h in crude extracts in Tris-HC1-cyanide buffer without any addition, prepared from thallus samples floated for 6 h at 26OC in the dark on nitrate-containing media. Values of specific activity were 0.21 and 0.19 m u , respectively. When thallus fragments are incubated on 10 mM nitrate in continuous darkness after a previous irradiation with either red light for 10 min or 10 min red light immediately followed by 10 min of far-red light, at a photon flux rate of 25 pmol . m-' s-I, the increase of nitrate reductase activity is always linear. It occurs at a constant rate of about 7 after red lighting and about 3 p U h-' after red + far-red irradiation, although statistically, there is no significant difference between these values (p < 0.05). When these irradiation programs were repeated at the start of each hour of incubation, reversal by

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CAN. J. BOT. VOL. 63, 1985


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I

"

0 0

1

2

3 4 5 6 Time ( hours)

7

Time ( hours)

FIG. 4. Time course of nitrate reductase activity in E. prunastri thallus floated on 10 mM KNO, when the incubations are started by ( 0 ) 10 min R, (0)10 min R 10 min FR, or when both treatments, ( 0 ) R and ( H ) R + FR, are repeated at the beginning of each hour of incubation. The photon flux rate was always 25 pmol - m-' s-I. Data are the means of three replicates. Vertical bars give standard error where it is larger than the symbols.

+

FIG. 5. Time course of nitrate reductase activity in isolated symbiont~from thalli maintained in continuous darkness ( 0 , ,). repeated red light ( 0 , H ) , or repeated R + FR light ( A , A). Open symbols show the activity of the photobiont and solid symbols that of the mycobiont. Data are the means of four replicates. Vertical bars give standard error where it is larger than the symbols. Photon flux rate was always 250 pmol - m-' s-I.

TABLE1. Reversibility and reciprocity effects of both red and far-red light on nitrate reductase activity and PI, content of Evernin prunnstri

A%

A&r

0.087*0.007

0.002*0.001

0.03

0.064*0.006 0.05120.004 0.015*0.002 0.02620.003

0.061 0 . 0 0 6 0.04420.004 0.016*0.002 0.017*0.002

0.49 0.46 0.52 0.38

Treatment Continuous darkness

+ + + +

R (10 min) darknesst R (10 min) FR (10 min) darknesst R (I min) darkness$ R (1 min) FR (1 min) darkness$

+

+

+:N

Rate of increase of nitrate reductase activity (pU . h-') At the start, 30.0*2.0; at the end, 0.22k0.03 30.0&2.0 25.0k2.0 36.02 1.5 20.0*2.0

NOTE: AA,, change in absorbance level following red-light treatment; AA,, change in absorbance level following red + far-red light treatment; m, far-red light. *Estimation of 4 values was carried out by using suspensions o l broken photobiont cells isolated after light treatments. 4 was calculated as

R, red light;

AA,/4

+

AAv

tPhoton flux rate was 25 pmol . m-'. s-I at the start of incubation. Correlation coefficient betwcen 4 and activity values, 0.027; standard error. 0.01; p < 0.05. $Photon flux rate was 250 pmol m-' sC' at the start of incubation. Correlalion coefficient between ~b and activity values, 0.078; standard error. 0.03; p < 0.05.

.

.

far-red light of the effect of red irradiation becomes more evident. The rate of increase of enzyme activity is 11 kU h-' after a repeated red irradiation program, whereas a value of about 4.5 kU h-' is achieved after a repeated irradiation with red + far-red light (Fig. 4). The reciprocity principle of light action, as it has been established by Kadota et al. (1979) by studying the effect of blue or far-red light on the elongation of Adianthum capillus-veneris protonemata, is also followed since, as it is shown in Table 1, the action of both red or red + far-red light, at a photon flux rate of 250 kmol m-' s-' for 1 min, or nitrate reductase activity is almost identical with that obtained by using a photon flux rate 10 times lower for 10 min. In addition, these results are closely proportional to Pi, content, estimated in fragments of algal cells, broken by sonic oscillation (also shown in Table 1). The correlation coefficient between photoequilibrium and nitrate reductase activity values indicates that the relation between both parameters is significant (p < 0.05). This implies

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that Pi, has a positive effect on nitrate reductase activity in E. prunastri. Analysis of data on enzyme activity in algae and fungi, recently obtained from thalli incubated for 4 h in the dark on 10 mM K N 0 3 , after a pulse of red light, indicates that the enzyme is equally distributed between both symbionts (Table 2). However, it is necessary, at this stage, to determine whether the action of light is exerted on both symbionts, in spite of the absence of previous literature about fungal phytochrome, or whether the contribution of algal cells on total nitrate reductase activity of whole thallus is the sole mechanism affected by light. The time course of enzyme activity of isolated symbionts from thalli irradiated with a photon flux rate of 250 kmol m-2 . s-' for 10 min each hour is shown in Fig. 5. The activity of algal nitrate reductase increases after red light treatment, but this effect is reversed by far-red light. Fungal nitrate reductase is not affected by any light treatment, but it is activated during incubation of thaIlus samples in the dark.

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AVALOS A N D VICENTE

TABLE 2. Location of nitrate reductase activity in the symbionts of Evernia prunastri


Specific activity

Total activity?

Symbiont*

pU

%

PU

%

Photobiont Mycobiont

6.5r0.7 4.4k0.5

60.0 40.0

49.425.0 50.125.0

50.0 50.0

*Symbionts were isolated from thalli floated on 10 mM nitrate in the dark at 26°C for 4 h with an initial R pulse of 25 pmol . m-' s-' for 10 min. tThe ratio protein of mycobiont/protein of phycobiont was 1.5.

.

Discussion The lichen Evernia prunastri possesses nitrate reductase activity when the thalli are floated on nitrate solutions. This fact would indicate a possible induction of the enzyme by its substrate, as has been reported for green alga (Diez et al. 1977; Guerrero et al. 1981) or free-living fungus (Rivas et al. 1974). Seemingly, Evernia nitrate reductase is also induced by nitrate, since the increase of activity in the dark, shown in Fig. 3, requires continuous synthesis of protein, as is indicated by the nearly complete dissappearance of this activity when 100 p M cycloheximide is added to the inducer-containing medium. In addition to this fact, logarithmic kinetics describing the relationship of increase of enzyme activity and time of incubation would indicate that the nitrate reductase induction depends upon the inducer availability. The effect of nitrate on the appearance of nitrate reductase activity is slightly enhanced when the thalli are irradiated with red light for the first 10 min incubation. This enhancement is slightly increased the more often the light treatment occurs. Therefore, 10 min red light at the beginning of each hour incubation gives a rate of increase of enzyme activity 1.6 times higher than that obtained after only one initial lighting. This fact possibly implies a high irradiance reaction, since even the reversal of the red light effect by far-red light is only significant after the repeated program of irradiation, i.e., when red plus far-red light is given 7 times through all the incubation time. These results confirm the action of PI, as having a positive effect on nitrate reductase in E. prunastri. In addition, the action of red light and, even, the effect of red plus far-red treatment follow the reciprocity law (Table 1) since very similar values of increase of enzyme activity are reached when the treatments use a photon flux rate of 25 pmol m-' s-' for 10 min or 250 pmol m-' s-' for 1 min. This principle can be taken as sound proof of the involvement of P,, in the enzyme induction, as demonstrated by Kadota et al. (1979) in the Prr-mediated elongation of A. capillus-veneris protonemata. Table 1 shows that the increase of nitrate reductase activity is a direct function of the percent of P,, actually present in suspensions of algal cells recently isolated from thallus samples which have received different light regimes. This relationship between Prrin the photobiont cells and nitrate reductase activity in the thallus is statistically significant and confirms the absence of phytochrome in fungi (Gressel and Rau 1983). These conclusions are in agreement with the analysis of enzyme activity of symbionts isolated from thalli floated on 10 mM nitrate in the dark or following different light treatments (Fig. 5). According to these data, enzyme induction in the dark is achieved by the mycobiont cells since these are the sole partner in which nitrate reductase increases with the time of incubation (the low activity found in the photobiont dissappears

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after 2 h incubation). However, red light only modifies the activity of algal nitrate reductase by increasing it and this enhancement is prevented by exposure to far-red light after red irradiation. Figure 5 contains some additional information since the level of nitrate reductase activity found in isolated symbionts is about 100 times higher than that found in the whole thallus. Isolation of the partners seems to be a partial purification of the enzyme. This fact can be explained on the basis of a release of exocellular proteins to the buffer in which both symbionts are isolated. The recovery of total protein from the symbionts is four times lower than that obtained by extracting whole thalli. These secreted proteins do not include nitrate reductase, which is restricted to the chloroplast (Guerrero et al. 1981) in higher plants or to the periplasmic space of phototrophic bacteria (Sawada and Satoh 1980); many of the other secreted proteins are related to the attachment of this lichen to its substrate (Yague et al. 1984) or to the exogenous use of very different nitrogen sources (Blanco et al. 1984; Legaz 1985). The consequence of the loss of an amount of protein other than nitrate reductase implies a rise of that protein's specific activity.

Acknowledgement This work was supported by a grant from the Comisi6n Asesora Cientifica y Tkcnica (Spain), No. 1733-82. BAREA, J. L., F. SOSA,and J. CARDENAS. 1976. Cyanide inactivation of Chlamydomonas reinhardi nitrate reductase under reducing conditions. Z. Pflanzenphysiol. 79: 237 -245. BLANCO, M. J . , C. SUAREZ, and C. VICENTE. 1984. The use of urea bv Evernia ~runastrithalli. Planta. 162: 305-310. B U ~ E R ,W. L., and K. H. NORRIS.1960. The spectrophotometry of dense high-scattering material. Arch. Biochem. Biophys. 87: 31 -40. DIEZ,J., A. CHAPARRO, J. M. VEGA,and A. RELIMPIO. 1977. Studies on the regulation of assimilatory nitrate reductase in Ankistrodesmus brownii. Planta, 137: 231 -234. ESTEVEZ, M. P., M. E. LEGAZ,L. OLMEDA, F. J. PEREZ,and C. VICENTE. 1981. Purification and properties of a new enzyme from Evernia prunastri, which reduces L-usnicacid. Z. Naturforsch. Teil C, 36: 35-39. FAHSELT, D. 198 1. Lichen products of Cladonia stellaris and Cladonia rangiferir~amaintained under artificial conditions. Lichenologist, 13: 87-91. GANDHI, A . P., and M. S. NAIR.1974. Role of roots, hormones and light in the synthesis of nitrate reductase and nitrite reductase in rice seedlings. FEBS Lett. 40: 343-345. GILES,K. L. 1970. The phytochrome system, phenolic compounds and aplanospore formation in a lichenized strain of Trebouxia. Can. J. Bot. 48: 1343- 1346. GRESSEL, J., and W. RAU. 1983. Photocontrol of fungal development. In Photomorphogenesis. Part B. Edited by W. Shropshire and H. Mohr. Springer-Verlag, Berlin, Heidelberg, New York, and Tokyo. pp. 603-639. GUERRERO, M. G., J. M. VEGA,and M. LOSADA. 1981. The assimilatory nitrate-reducing system and its regulation. Annu. Rev. Plant Physiol. 32: 169-204. HARTREE, E. F. 1972. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48: 423-427. JOHNSON, C. B. 1976. Rapid activation by phytochrome of nitrate reductase in the cotyledons of Sinapis alba. Planta, 128: 127- 131. JONES,R. W., and R. W. SHEARD. 1972. Nitrate reductase activity: phytochrome mediation of induction in etiolated peas. Nature (London) New Biol. 238: 221 -222. KADOTA, A., M. WADA,and M. FURUYA. 1979. Apical growth of


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